Process for the isolation of a pharmaceutically acceptable alkali metal salt of clavulanic acid

ABSTRACT

The invention relates to the process for the isolation of a pharmaceutically acceptable alkali metal salt of clavulanic acid from a fermentation broth containing impure clavulanic acid comprising the steps of filtration of the fermented broth, extraction of the clavulanic acid to a water immiscible or partly water immiscible solvent at pH from 1.2-2, precipitation of an alkali metal salt A of clavulanic acid by addition of a solution of an alkali metal alkylalkanoate, characterized by the following steps: 
     before the filtration the fermented broth containing clavulanic acid is diluted with water, a flocculating agent is added and the pH is adjusted to 3-5 
     for further purification the alkali metal salt A of clavulanic acid is converted to clavulanic acid by addition of an inorganic acid and is extracted into a water immiscible or partly water immiscible solvent 
     a solution of a different alkali metal B alkyl alkanoate is added and the alkali metal salt B of clavulanic acid is precipitated.

This application is filed under U.S.C. §371 from PCT/EP98/01637.

The present invention relates to a novel process for the isolation ofthe pharmaceutically acceptable alkali metal salt of clavulanic acidfrom a fermentation broth containing impure clavulanic acid comprisingthe steps of filtration of the fermented broth, extraction of clavulanicacid to a water immiscible or partly water immiscible solvent at a pHfrom 1.2 to 2, precipitation of the alkali metal salt A of clavulanicacid by addition of a solution of an alkali metal alkylalkanoate.

Clavulanic acid and its alkali metal salts and esters are used inpharmaceutical preparation to prevent the deactivation of β-lactamantibiotics. Commercial preparations of clavulanic acid containpotassium clavulanate in combination with amoxycillin trihydrate.Clavulanic acid is an unstable hygroscopic oil. Potassium clavulanate ismore stable than the free acid or other salts, and is therefore mostfrequently, used for commercial preparations.

Clavulanic acid and its derivatives are inhibitors of the β-lactamases.The resistance of β-lactam antibiotics is associated with inactivationof β-lactam structure due to the opening of β-lactam ring by β-lactamaseproduced by bacteria. Thus the inactivating enzymes are commonly calledas β-lactamase, they are divided into penicillinase andcephalosporinase.

Furthermore clavulanic acid itself is believed to have an antibacterialactivity.

Clavulanic acid is produced from various strains of microorganism by afermentation process. For this process for example strains belonging tothe genus Streptomyces such as S. clavuligerus NRRL 3585 (U.S. Pat. No.4,110,165), S. jumonjinensis NRRL 5741 (British Patent 1,563,103), S.katsurahamanus IFO 13716 (Japanese Patent 83,009,579), and Streptomycessp. P6621 FERM 2804 (Japanese Patent 55,162,993) are used. For thepreparation of clavulanic acid by a fermentation process themicroorganism Streptomyces clavuligerus is preferred.

In the state of art different processes for the preparation andpurification of the clavulanic acid containing fermentation broth aredescribed.

GB 1 508 977 discloses preparation of clavulanic acid and its salts byfiltration of the fermentation broth by passage through an anionicexchange resin.

GB 1 543 563 discloses a fermentation process wherein the pH value ofthe medium is maintained in the range of 6.3 to 6.7. A pharmaceuticallyacceptable salt such as potassium clavulanate is prepared by ionexchange process from lithium clavulanate. However lithium clavulanateis not a pharmaceutically acceptable salt. Therefore it is necessary toadd an ion exchange process as a further step for preparing apharmaceutically acceptable form of the compound. Furthermore, theremaining salt lithium chloride is soluble in organic solvents andtherefore it is difficult to separate the lithium chloride in theaqueous phase during the extraction process.

Further documents of the state of the art like EP-0 647 229 describe theuse of amine salts of clavulanic acid as intermediate compounds for thepreparation and purification of clavulanic acid and its alkali metalsalts. EP-0 647 229 for example describes a process for the preparationof a purified clavulanic acid or a salt or ester thereof by preparing adiamine salt of clavulanic acid and converting this intermediatecompound into clavulanic acid or a pharmaceutically acceptable salt orester. The conversion is made by adding for example potassium2-ethylhexanoate and precipitating potassium clavulanate. Many of suchamines are either unsuitable for the production of a salt of clavulanicacid or they give rise to amine salts of clavulanic acid which areeither hygroscopic or toxic or both and, therefore, are unsuitable foruse as intermediates for the preparation of a pharmaceuticallyacceptable compound.

Other purification processes of the state of the arts are performedwithout any amine compounds. For example WO 95/34194 A2 describes aprocess for manufacturing an alkali metal salt of clavulanic acidwherein impure clavulanic acid in aqueous solution is extracted by asolvent mixture of ketone and alkyl acetate under acidic condition. Thesolution is than treated in a conventional manner and the solution of analkali metal salt of alkanoic acid dissolved in ketone or alkanolsolvent is added to obtain pure alkali metal salt of clavulanic acid.Thus the process according to this state of the art omits the step offormation of amine salts. This process has the advantage that the use ofmostly toxic amines is no longer necessary. In a preferred embodiment asalkali metal salt of alkanoic acid sodium or potassium salts are used,especially potassium 2-ethylhexanoate.

A similar process is also described in WO 96/28452 A1. This processcomprises the steps of removing solids from a clavulanic acid containingfermentation broth by microfiltration, acidifying the filtrate to a pHbetween 1 and 3, extracting the acidified filtrate with a waterimmiscible solvent and separating the clavulanic acid containingextract. This extract is mixed with a metal donor and at least oneadditional solvent. From the solution the metal clavulanate salt isseparated.

As metal donor compounds organic salts, carbonates, bicarbonates orhydroxides of potassium, sodium, lithium or magnesium can be used. Theuse of carboxylic acid salt is preferred. Further preferred metal donorsinclude potassium 2-ethylhexanoate, potassium acetate, lithium2-ethylhexanoate and lithium acetate.

EP-0 182 522 B1 also describes a process for the preparation ofclavulanic acid and its salts and esters. In this process thefermentation broth is worked up as follows. The solids are removed byfiltration or centrifugation. The broth is acidified to a pH of 1 to 3and clavulanic acid is extracted by adding a water immiscible solventwith two phases being separated for example by centrifugation. Thisgives the clavulanic acid in the water immiscible phase. The solution ispurified by mixing it with the dissolved lithium 2-ethylhexanoatesolution isolating lithium clavulanate and optionally converting thelithium salt to other salts or an ester. The conversion of the lithiumsalt to other salts is carried out by ion exchange procedures using ionexchange resins in the form of the desired cation preferably sodium orpotassium.

The processes of the state of the art referred above have thedisadvantage, that the alkali metal salts prepared by directprecipitation are not pure enough for a pharmaceutical use. Therefore,further purification steps like recrystallization, purification over acolumn etc. are necessary.

The object of the invention therefore is to prepare clavulanic acid andits pharmaceutically acceptable alkali metal salts such as potassiumclavulanate in a new and simple manner wherein the desired substance isobtained in a very high yield without any additional purification stepsand of high purity avoiding the use of toxic amines or lithiumcompounds.

This technical problem is solved by a process which is characterized bythe following steps:

before the filtration of the fermentation broth, the fermentation brothcontaining clavulanic acid is diluted with water, a flocculating agentis added and the pH is adjusted to pH 3 to 5,

for further purification the alkali metal salt A of clavulanic acid isconverted to clavulanic acid by addition of an inorganic acid and isextracted into a water immiscible or partly water immiscible solvent,

and to the solution of clavulanic acid a solution of a different alkalimetal alkylalkanoate B is added and the alkali metal salt B ofclavulanic acid is precipitated.

Suitable salts according to the present invention are pharmaceuticallyacceptable alkali metal salts and alkaline earth metal salts likesodium, potassium, calcium and magnesium salts.

Of these compounds potassium clavulanate is the most stable compoundwhich is normally used for pharmaceutical preparations. The clavulanicacid itself is an unstable hygroscopic oil which is not used for thepreparation of pharmaceutical compounds.

In a preferred embodiment the alkylalkanoate is an alkylhexanoate,especially 2-ethylhexanoate. The alkali metal A is sodium and the alkalimetal B is potassium.

In a further preferred embodiment the filtered broth containingclavulanic acid is purified after the filtration step by adsorption onan anion exchange resin containing column and eluted with an aqueoussolution of an alkali metal salt. As anionic exchange resin for exampleDIAION® SA-11A is used. After the elution step clavulanic acid isextracted into a water immiscible solvent or a partly water immisciblesolvent and sodium clavulanate is precipitated by the addition of sodium2-ethylhexanoate solution in an appropriate solvent after dehydrationwith anhydrous sodium or magnesium sulphates and purification withactivated carbon.

As an alternative process step it is also possible to carry out thedirect extraction of the clavulanic acid contained in the filtered brothto a water immiscible or partly water immiscible solvent at an adequatepH between 1.2-2.0 without using a column. Preferably 3 to 4 volumes ofsolvent in relation to the filtered broth are used in this step. Theclavulanic acid is extracted from the above solvent to an aqueoussolution using an organic base, preferably triethylamine ordiethylamine. In this operation, a concentration of about 10 to 15 timesis obtained. The exhaust solvent can be re-used in the process without apurification step. The clavulanic acid is back extracted to a waterimmiscible or partly water immiscible solvent. From the solvent sodiumclavulanate is precipitated by addition of the sodium 2-ethylhexanoatesolution in an appropriate solvent after dehydration.

Clavulanic acid is a hygroscopic oil and is not very stable in aqueoussolution. Therefore, this solution of clavulanic acid in a waterimmiscible or partly water immiscible solvent is dehydrated in apreferred manner by addition of anhydrous sodium or magnesium sulphateand further purified by addition of activated carbon. By the use of theactivated carbon the coloured impurities are removed from the solution.

In a preferred embodiment as solvent ethyl acetate, butyl acetate,methyl isobutyl ketone or mixtures thereof are used. Furthermore, beforethe filtration step a flocculating agent can be used. As flocculatingagents quaternary ammonium salts are preferred.

A further object of the present invention is the use of sodiumclavulanate as intermediate compound for the preparation of thepharmaceutically acceptable potassium clavulanate.

Filtration of clavulanic acid fermented broth is normally difficult andthe use of flocculating agents has been suggested to improvefilterability of the whole broth as for example described in EP-A 0 387178. The present invention describes a new simple and cheap method toimprove not only the filterability of the whole broth but also a meansof increasing the yield of filtration and a facilitation of thesubsequent down stream process.

Surprisingly it has been found possible to improve the recovery ofclavulanic acid from an aqueous fermented broth by pre-diluting it withwater. This pre-dilution of the fermented broth when combined with theuse of flocculating agents and pH-adjustment is even more efficient,both at the level of filterability and yield of filtration andsubsequent down stream operations. The pre-dilution of the broth withwater decreases the viscosity of the same making the filtration easier.The yield of filtration is also improved since a poorer filtrate remainsin the filter cake. The use of a flocculating agent and the gentleacidification to a pH value of 3 to 5 leads to precipitation of proteinswhich will be retained in the filter cake improving the filtration rate.In this way, a purer filtrate is obtained implying that the furtherrecovery operations for the clavulanic acid are much easier. Accordingto the present invention high quality potassium clavulanate can beproduced with good yields from the fermented broth of clavulanic acidproducing microorganisms through the steps which are described in thecharacterizing part of claim 1.

After filtration of the fermented broth the solution can be directlyextracted to an organic solvent at an adequate pH is about 1.2 to 2.0,preferably using 3 to 4 volumes of solvent in relation to the filteredbroth. It is also possible to adsorb the solution before that step ontoan anionic exchange resin and to elute clavulanic acid with an aqueoussolution of an alkaline metal salt. The extraction step leads to anorganic phase which contains the clavulanic acid from the fermentationprocess. The organic phase is dehydrated with anhydrous sodium ormagnesium sulphate and purified with activated carbon. Thereafter sodium2-ethylhexanoate in an organic solution is added and after a period ofcrystallization crystals of sodium clavulanate can be collected byfiltration.

The conversion from sodium clavulanate to potassium clavulanate iscarried out by extraction of clavulanic acid to an adequate solvent andcrystallization of potassium clavulanate after dilution of the acid withan adequate solvent and addition of potassium 2-ethylhexanoate orpotassium acetate solution in an appropriate solvent. For this processstep sodium clavulanate is suspended in a mixture of methyl isobutylketone or ethyl acetate or butyl acetate with water. To this solution aninorganic acid is added. By this addition the salt of the clavulanicacid is converted into clavulanic acid. The acid is extracted into theorganic phase with stirring.

The mixture is then diluted with isopropanol and a solution of potassium2-ethylhexanoate in isopropanol is added to reach a pH between 6 and 7.After a crystallization period of 2 hours at low temperature potassiumclavulanate crystals can be collected by filtration.

If necessary potassium clavulanate can be recrystallized.

In a further preferred embodiment it is possible to extract theclavulanic acid from the water immiscible or partly water immisciblesolvent to an aqueous solution using an organic phase preferablytriethylamine or diethylamine. By this operation concentration of about10 to 15 times can be obtained. The exhaust solvent can be reused in theprocess without any purification step.

A further alternative way is a back extraction of clavulanic acid to thewater immiscible or partly immiscible solvent.

By the process of the present invention clavulanic acid salts ofpotassium can be prepared in high purity and good yields. The process issimple and allows to work without the use of any toxic amines.Furthermore the purity of the potassium clavulanic acid prepared by thepresent invention is higher then the purity of this compound made fromprocesses of the state of the art which directly use the precipitationby reaction with potassium 2-ethylhexanoate.

The following examples are intended to illustrate the present not tolimit it.

A. Experiments of Filtration of Fermented Broth of Clavulanic Acid

EXAMPLE 1 Effect of Pre-dilution of Fermented Both with Water on theRate and Yield of Filtration

Portions of 200 ml of clavulanic acid fermented broth, to which 4% (w/v)of filter aid (Dicalite 478) was added, were water and filtered undervacuum in a Buckner filter.

The rates and yields of filtration obtained are shown in Table 1

TABLE 1 Percentage of pre- dilution with water Filtration rate Yield offiltration (v/v) (1 m⁻²h⁻¹) (%) 0 16 85.3 10 18 87.5 20 20 90.1 30 2591.6 40 30 92.5 50 35 92.6 60 30 92.8 70 28 93.5

EXAMPLE 2 Combined Effect of Fermented Broth Pre-dilution and Use ofFlocculating Agents on the Rate and Yield of Filtration

Portions of 200 ml of clavulanic acid fermented broth were treated 4 %(w/v) of filter aid (Dicalite 478) and with 0.4% (v/v) of Rolquat CDM-BCas flocculating agent. Each of the 200 ml portion was diluted with waterand filtered as described in example 1.

The rates of filtration obtained were as shown in Table 2. The yields offiltration were similar to those obtained in Example 1.

TABLE 2 Percentage of pre-dilution Filtration rate with water (v/v) (1m⁻²h⁻¹) 0 25 10 30 20 35 30 42 40 46 50 50 60 45 70 40

EXAMPLE 3 Combined Effect to Fermented Broth Pre-dilution with the Useof a Flocculating Agent and pH Adjustment to 4.5 on the Rate and Yieldof Filtration

Portions of 200 ml of clavulanic acid fermented broth were treated with4% (w/v) of filter aid (Dicalite 478) and with 0.4% (v/v) of theflocculating agent used in Example 2. Then, the pH of each of these 200ml portions of fermented broth was adjusted to pH 4.5 with 15 % (w/v)sulphuric acid. Each portion was diluted with water and filtered as inExample 1. The rates of filtration obtained are shown in Table 3. Theyields of filtration obtained were similar to those obtained in Example1.

TABLE 3 Percentage of pre-dilution Filtration rate with water (v/v) (1m⁻²h⁻¹) 0 50 10 55 20 60 30 62 40 70 50 75 60 70 70 65

B. Preparation of the Intermediate Salt of Clavulanic Acid

The following examples illustrate the preparation of the intermediatesalt of clavulanic acid (sodium clavulanate).

EXAMPLE 4

750 l of fermented broth of Streptomyces clavuligerus, assaying about3800 μg/ml (as clavulanic acid), were treated with 4% (w/v) of filteraid, prediluted with 40 % of deionised water and filtered in a pressfilter after the addition of 0.4% (v/v) of a flocculating agent (RolquatCDM BC) and after the adjustment of the pH to 4.5 with 15% sulphuricacid (w/v). After the filtration, the cake was washed with 10% (v/v) ofwater leading to a total volume of filtered broth of about 1100 l, withan assay of 2500 μg/ml (yield of filtration about 92%).

The filtrate containing the clavulanic acid was adsorbed onto an anionicresin (Diaion SA-11A) in columns and the adsorbed clavulanic acid elutedwith a solution of a 1.0M sodium chloride. The combined yield ofadsorption/elution was approximately 85% of the theoretical and theaverage concentration of the mixed rich eluate obtained was about 10000μg/ml. This eluate was extracted with 4 volumes of ethyl acetate at pHaround 1.4 with 25% (w/v) sulphuric acid to give an ethyl acetateextract with a concentration of about 2000 μg/ml. Butyl acetate ormethyl isobutyl ketone can also be used. The ethyl acetate extract wastreated with anhydrous sodium sulphate and activated carbon and filteredusing a Buckner type filter. The filtrate obtained was again treatedwith anhydrous sodium sulphate and filtered.

From the dehydrated, purified ethyl acetate extract the intermediatesodium clavulanate was obtained as follows:

A 0.3 M solution of sodium 2-ethylhexanoate in ethyl acetate wasprepared. This solution was treated with anhydrous sodium sulphate andactivated carbon and filtered. Afterwards, this clear solution was addedto the ethyl acetate extract, for one hour, to reach a pH within therange 6.0-6.2. After this addition, and after a period ofcrystallization, of approximately one hour at low temperature (5° C.),the crystals of sodium clavulanate were collected by filtration(centrifugation can also be used) washed two times with acetone anddried with a stream of nitrogen gas under vacuum. The analysis of thesodium clavulanate was 70% (as clavulanic acid) meaning a global yieldfrom the whole broth of about 58% of the theoretical.

EXAMPLE 5

The intermediate salt was prepared as in Example 1 with the followingdifferences:

The solvent used to extract clavulanic acid from the mixed rich eluatewas butyl acetate which was dehydrated with anhydrous sodium sulphateand purified with activated carbon. After solids removal, purified butylacetate was treated with a 0.3 M solution of sodium 2-ethylhexanoate inethyl acetate. The crystallization of the sodium clavulanate waseffected during 1.5 hours at 5° C. The analysis of the sodiumclavulanate was about 70% as clavulanic acid and the yield from thewhole broth about 45% of the theoretical.

EXAMPLE 6

The intermediate salt was obtained as in Example 1 with the followingdifferences:

The solvent used to extract clavulanic acid from the rich eluate wasmethyl isobutyl ketone which after extraction was dehydrated withanhydrous sodium sulphate and purified with activated carbon. Aftersolids removal, the purified methyl isobutyl ketone containingclavulanic acid was treated with a solution of a 0.3M sodium2-ethylhexanoate in methyl isobutyl ketone. The crystallization ofsodium clavulanate was effected during 1.5 hours at 5° C. The analysisof the sodium clavulanate was about 70% as clavulanic acid and the finalyield from the whole broth about 50% of the theoretical.

EXAMPLE 7

The intermediate salt was obtained as in Example 1 with the followingdifferences:

Filtered broth was extracted directly to ethyl acetate. Afterwardsclavulanic acid was extracted to an aqueous solution usingtriethylamine. Clavulanic acid in the aqueous solution was backextracted to the same solvent and the crystallization on of sodiumclavulanate was performed using a 0.3 M solution of sodium2-ethylhexanoate in ethyl acetate.

The analysis of the sodium clavulanate obtained was 72% (as clavulanicacid) and the global yield obtained from the whole broth wasapproximately 50% of the theoretical.

C. Preparation of Pure Potassium Clavulanate

EXAMPLE 8

1.5 kg (as clavulanic acid) of intermediate sodium clavulanate, preparedaccording to the procedures described in part B Examples 4 to 7, weresuspended in a mixture of methyl isobutyl ketone and deionised water(97/3, v/v) at a temperature of 5° C. Diluted pure hydrochloric acid wasadded to reach pH 1.3 and the clavulanic acid was extracted to theorganic phase with stirring. After phase separation by gravity, the richmethyl isobutyl ketone was dehydrated with anhydrous sodium sulphate (80g/l) at low temperature. The sodium sulphate was removed by filtrationand the filtrate obtained was treated again with more anhydrous sodiumsulphate (20 g/l) and decolourized with activated carbon. Afterfiltration, using diatomite as filter aid, the rich solvent obtained wasdiluted with isopropanol.

To the mixture thus obtained, a 0.3M solution of potassium2-ethylhexanoate in isopropanol was added for 1.5 hours, to reach a pHaround 6.5. After the addition of the potassium 2-ethylhexanoatesolution and a crystallization period of 2 hours at low temperature, thepotassium clavulanate crystals were collected by filtration in a closedfilter, washed with acetone, pressed with a nitrogen stream and driedunder vacuum at room temperature to a moisture level below 0.5%. Theassay of the product obtained was 82% (as clavulanic acid). Theconversion yield from sodium clavulanate was 80% of the theoretical.

EXAMPLE 9

1.5 kg (as clavulanic acid) of intermediate sodium clavulanate weresuspended in a mixture of butanol/water (90/10, v/v) and diluted purehydrochloric acid was added until pH 1.3, keeping the temperature below5° C. The sodium salt of clavulanic acid was converted into clavulanicacid and extracted to the solvent. The aqueous phase was separated bygravity. After phase separation, the water in the solvent was removed byazeotropic distillation at reduced pressure, and the rich concentratedsolvent was treated with activated carbon and filtered. To the richpurified filtered solvent, a 0.3M solution of potassium 2-ethylhexanoatein butanol was added for 1.5 hours, at room temperature.

After the addition of the potassium 2-ethylhexanoate solution, thereaction mixture was cooled to 5° C. and after a crystallization periodof 1.5 hours, the potassium clavulanate crystals were collected byfiltration in a closed filter, washed with acetone, pressed with anitrogen stream, and dried under vacuum at room temperature to amoisture level below 0.5%.

The assay of the product obtained was 81% (as clavulanic acid). Theconversion yield from sodium clavulanate was 75% of the theoretical.

EXAMPLE 10

1.5 kg (as clavulanic acid) of intermediate sodium clavulanate preparedaccording to the procedures of part B examples 4 to 7, were suspended ina mixture of butyl acetate/water (97/3, v/v) and diluted purehydrochloric acid was added until pH 1.3. The temperature was kept below5° C. The sodium salt of clavulanic acid was converted into clavulanicacid and extracted to the solvent. After extraction and phaseseparation, the rich butyl acetate phase was treated with anhydroussodium sulphate and activated carbon, and filtered. The rich filteredsolvent obtained was diluted with isopropanol. To the mixed solventobtained, a 0.3M solution of potassium 2-ethylhexanoate in isopropanolwas added for 1.5 hours, to reach a pH around 6.5. After acrystallization period of 2 hours, at 5° C., the crystals of potassiumclavulanate were recovered by filtration in a closed filter, washed withacetone, pressed with a nitrogen stream, and dried under vacuum at roomtemperature to a moisture level lower than 0.5%. The conversion yieldfrom sodium clavulanate was 78% of the theoretical.

EXAMPLE 11 Recrystallization of Potassium Clavulanate

When required potassium clavulanate can be purified as described in theexample below. A mixture of methyl isobutyl ketone and water (98/2,v/v), was prepared. Potassium clavulanate was suspended in thepreviously prepared mixture, and diluted pure hydrochloric acid wasadded until pH 1.3. The temperature was kept below 5° C. The potassiumclavulanate was converted into clavulanic acid and extraced to thesolvent. After extraction and phase separation, the rich methyl isobutylketone was treated with anhydrous sodium sulphate and activated carbon,and filtered. Isopropanol was added to the methyl isobutyl ketone and a0.3M solution of potassium 2-ethylhexanoate in isopropanol was added for1 hour at room temperature. After a crystallization period of 3 hours at5° C., the crystals of potassium clavulanate were recovered byfiltration in a closed filter, washed with anhydrous acetone, pressedwith a nitrogen stream and dried under vacuum at room temperature, toreach a moisture level lower than 0.5%. The yield of recrystallizationwas about 80% of the theoretical.

What is claimed is:
 1. A method for isolating a pharmaceuticallyacceptable alkali metal salt of clavulanic acid from a fermentationbroth comprising clavulanic acid, comprising: a) diluting a fermentationbroth comprising clavulanic acid by adding water in a volume equal tobetween ten percent and seventy percent of the volume of thefermentation broth, resulting in a diluted fermentation broth comprisingclavulanic acid; b) filtering the diluted fermentation broth of a),resulting in a filtered fermentation broth comprising clavulanic acid;c) extracting the clavulanic acid to a water immiscible or partly waterimmiscible solvent, resulting in a solution comprising clavulanic acid;d) adding a solution of a first alkali metal alkylalkanoate to thesolution of c), and precipitating an alkali metal salt A of clavulanicacid; e) converting the alkali metal salt A of clavulanic acid toclavulanic acid by adding an inorganic acid; f) extracting theclavulanic acid to a water immiscible or partly water immisciblesolvent; and g) adding a second alkali metal alkylalkanoate andprecipitating an alkali metal salt B of clavulanic acid.
 2. The methodof claim 1, wherein the alkali metal A is sodium and the alkali metal Bis potassium.
 3. The method of claim 1, wherein, prior to extracting theclavulanic acid to a water immiscible or partly water immisciblesolvent, c) further comprises: i) adsorbing the clavulanic acid of b) onan anion exchange resin containing column, and ii) eluting theclavulanic acid adsorbed onto the anion exchange resin containing columnwith an aqueous solution comprising an alkali metal salt, resulting inan eluate comprising an alkali metal salt of clavulanic acid; whereinthe extraction in c) comprises adjusting the pH of the eluate to a valueeffective to convert the alkali metal salt of clavulanic acid in ii) toclavulanic acid and combining the eluate with the water immiscible orpartly water immiscible solvent.
 4. The method of claim 1, wherein c)further comprises dehydrating the water immiscible or partly waterimmiscible solvent containing the clavulanic acid.
 5. The method ofclaim 4, wherein dehydrating comprises adding anhydrous sodium sulfateor anhydrous magnesium sulfate to the solvent.
 6. The method of claim 1,wherein c) further comprises adding activated carbon to the waterimmiscible or partly water immiscible solvent containing the clavulanicacid.
 7. The method of claim 1, wherein, after extracting the clavulanicacid to the water immiscible or partly water immiscible solvent in c),c) further comprises: i) combining the solution comprising clavulanicacid in c) with an aqueous solution and an organic base under conditionseffective to cause extraction of a salt of clavulanic acid to theaqueous solution, and ii) combining the aqueous solution of i) with awater immiscible or partly water immiscible solvent under conditionseffective to cause extraction of clavulanic acid to the aqueoussolution; wherein the volume of water immiscible or partly waterimmiscible solvent in c) is between three and four times the volume ofthe filtered fermentation broth.
 8. The method of claim 1, wherein thewater immiscible or partly water immiscible solvents each are selectedfrom the group consisting of ethyl acetate, butyl acetate, methylisobutyl ketone, or any combination thereof.
 9. The method of claim 1,wherein, after diluting the fermentation broth, a) further comprisescombining a flocculating agent with the diluted fermentation broth. 10.The method of claim 9, wherein, after diluting the fermentation brothand prior to combining a flocculating agent with the dilutedfermentation broth, a) further comprises adjusting the pH of thefermentation broth to between 3 and
 5. 11. The method of claim 9,wherein the flocculating agent comprises a quaternary ammonium salt. 12.The method of claim 10, wherein the flocculating agent comprises aquaternary ammonium salt.
 13. The method of claim 1, wherein, afterextracting the clavulanic acid to the water immiscible or partly waterimmiscible solvent in c), c) further comprises: i) combining thesolution comprising clavulanic acid in c) with an aqueous solution andan organic base under conditions effective to cause extraction of a saltof clavulanic acid to the aqueous solution, and ii) combining theaqueous solution of i) with a water immiscible or partly waterimmiscible solvent under conditions effective to cause extraction ofclavulanic acid to the water immiscible or partly water immisciblesolvent.
 14. The method of claim 1, wherein the alkylalkanoate in d) isan alkylhexanoate.
 15. The method of claim 1, wherein the alkylalkanoatein d) is 2-ethylhexanoate.
 16. The method of claim 1, wherein the volumeof water added to the fermentation broth in a) is between about fortypercent and about sixty percent of the volume of the fermentation broth.17. The method of claim 1, wherein the alkylalkanoate in g) is analkylhexanoate.
 18. The method of claim 1, wherein the alkylalkanoate ing) is 2-ethylhexanoate.
 19. The method of claim 13, wherein the organicbase is triethylamine or diethylamine.
 20. The method of claim 1,wherein f) further comprises dehydrating the water immiscible or partlywater immiscible solvent containing the clavulanic acid.
 21. The methodof claim 20, wherein dehydrating comprises adding anhydrous sodiumsulfate or anhydrous magnesium sulfate to the solvent.
 22. The method ofclaim 1, wherein f) further comprises adding activated carbon to thewater immiscible or partly water immiscible solvent containing theclavulanic acid of f) prior to adding a solution of a second alkalimetal alkylalkanoate to the solvent of f).
 23. The method of claim 7,wherein the organic base is triethylamine or diethylamine.